Coupled Lipidomics and Digital Pathology as an Effective Strategy to Identify Novel Adverse Outcome Pathways in Eisenia fetida Exposed to MoS₂ Nanosheets and Ionic Mo

Molybdenum disulfide (MoS₂) nanosheets are increasingly applied in several fields, but effective and accurate strategies to fully characterize potential risks to soil ecosystems are lacking. We introduce a coelomocyte-based in vivo exposure strategy to identify novel adverse outcome pathways (AOPs) and molecular endpoints from nontransformed (NTMoS₂) and ultraviolet-transformed (UTMoS₂) MoS₂ nanosheets (10 and 100 mg Mo/L) on the earthworm Eisenia fetida using nontargeted lipidomics integrated with transcriptomics. Machine learning-based digital pathology analysis coupled with phenotypic monitoring was further used to establish the correlation between lipid profiling and whole organism effects. As an ionic control, Na₂MoO₄ exposure significantly reduced (61.2-79.5%) the cellular contents of membrane-associated lipids (glycerophospholipids) in earthworm coelomocytes. Downregulation of the unsaturated fatty acid synthesis pathway and leakage of lactate dehydrogenase (LDH) verified the Na₂MoO₄-induced membrane stress. Compared to conventional molybdate, NTMoS₂ inhibited genes related to transmembrane transport and caused the differential upregulation of phospholipid content. Unlike NTMoS₂, UTMoS₂ specifically upregulated the glyceride metabolism (10.3-179%) and lipid peroxidation degree (50.4-69.4%). Consequently, lipolytic pathways were activated to compensate for the potential energy deprivation. With pathology image quantification, we report that UTMoS₂ caused more severe epithelial damage and intestinal steatosis than NTMoS₂, which is attributed to the edge effect and higher Mo release upon UV irradiation. Our results reveal differential AOPs involving soil sentinel organisms exposed to different Mo forms, demonstrating the potential of liposome analysis to identify novel AOPs and furthermore accurate soil risk assessment strategies for emerging contaminants.